European Journal of Soil Biology 74 (2016) 1e8

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European Journal of Soil Biology

journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Effect of biochar additions to soil on nitrogen leaching, microbial

biomass and bacterial community structure
Nan Xu a, Guangcai Tan a, Hongyuan Wang b, *, Xiapu Gai b
a
Key Laboratory for Heavy Metal Pollution Control and Reutilization, School of Environment and Energy, Peking University Shenzhen Graduate School,
Shenzhen 518055, China
b
Key Laboratory of Nonpoint Source Pollution Control, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy
of Agricultural Sciences, Beijing 100081, China

a r t i c l e i n f o a b s t r a c t

Article history: Previous studies already demonstrated that biochar addition reduces nitrogen (N) leaching in soil, but
Received 22 July 2015 little information is available about its effects on N leaching and bacterial community structure under the
Received in revised form application of organic N. This study investigated the effects of corn-straw biochar under the application
16 February 2016
of urea (250 kg N ha
1) in layered soil columns. The PCR-amplified partial 16S rRNA genes in soil were
Accepted 17 February 2016
Available online xxx
sequenced before and after biochar treatment in order to assess the change of bacterial diversity and
community structure utilizing the Illumina technology. With the application of 2% (B2), 4% (B4) and 8%
Handling Editor: C.C. Tebbe (B8) biochar (mass ratio), the cumulative amount of total leached nitrogen was reduced by 18.8%, 19.5%
and 20.2%, respectively (P < 0.05). More than 90% of the total nitrogen leaching was in the form of nitrate,
Keywords: and increasing amount of biochar resulted in reduced amount of N leaching. The water holding capacity,
Biochar microbial biomass, pH, electrical conductivity, net N mineralization and respiration rate of the soil were
Nitrogen leaching all increased under biochar treatments, except that the B8 treatment decreased soil respiration rate and
Microbial biomass net N mineralization in comparison with B4. Bacterial diversity increased in biochar-amended soil and
Bacterial community structure
was positively correlated with the addition ratio of biochar. Dominant phyla across all samples were
High-throughput sequencing
Proteobacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Nitrospirae and Gemmatimonadetes.
The relative abundance of Acidobacteria, Chloroflexi and Gemmatimonadetes decreased under biochar
treatments, while that of Proteobacteria, Bacteroidetes and Actinobacteria increased. Overall, biochar
increased water holding capacity, enhanced microbial biomass and changed bacterial community
structure of the soil which may all have contributed to the reduction of nitrogen leaching.
© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction mitigation and positive soil amendment [3,4]. Biochar is a solid

Excessive and/or unbalanced application of nitrogen fertilizers
has caused the translocation of nitrogen (N) from farmlands into
aquatic systems. Nitrogen, especially in the form of nitrate, is easily
soluble in soil pore water, and readily infiltrates beneath the active
soil layer with crop root. The N leaching may deplete soil fertility,
accelerate soil acidification and reduce crop yields [1]. Moreover, N
leaching is regarded as a major contributor to the eutrophication of
surface and ground water [2].
Recently, the interest in applying biochar in soil has grown,
which is due to the dual benefits of biochar on both climate change
* Corresponding author.
E-mail address: wanghongyuan@caas.cn (H. Wang).
carbon-rich organic material generated by heating biomass under
condition of limited or no oxygen [5]. Previous studies already
demonstrated that biochar addition reduces N leaching. This could
be attributed to the increase of cation and anion exchange capac-
ities (CEC, AEC) of soil by the biochar material [6,7]. Another reason
could be the physical retention of available N dissolved in the soil
solution, as the water holding capacity also increased in biochar-
amended soil [6]. Although organic N, like urea, is widely used in
agriculture, little information is available about the effect of biochar
on N leaching and biological interactions under the application of
organic N.
Soil amendment with biochar could modify physical and
chemical properties of the habitat for microbial colonization, and
therefore affect soil microbial activity and community structure
[8,9]. Transformation of microbial communities can be associated

http://dx.doi.org/10.1016/j.ejsobi.2016.02.004
1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
2 N. Xu et al. / European Journal of Soil Biology 74 (2016) 1e8
with the change of nutrient turnover and utilization after the
addition of biochar [10]. The carbon-rich “Amazonian dark soils”
(Arthrosols) are evidence, which host distinct microbial commu-
nities in comparison with adjacent carbon-poor soil, and have
higher microbial biomass and diversity as well [11]. Various
methods have been used to investigate the microbial communities
in biochar-amended soils. The bacterial community composition in
Brazilian anthrosols and adjacent soils was investigated by using
traditional culturing [11]. Some studies adopted phospholipid fatty
acids analysis to describe soil microbial communities responding to
biochar [12,13]. PCR-denaturing gradient gel electrophoresis
method was also widely deployed to analyze the changes of mi-
crobial structure under the addition of biochar [14,15]. However,
most of these studies were conducted under a constant incubation
condition, instead of the simulated N leaching condition which
would be better. Also, the previous studies have only considered
the dominant microbial taxa, while next-generation DNA-
sequencing technologies of PCR products now offer the potential to
also detect less abundant taxa and thus giving a more complete
picture of the microbial communities [16].
The main objectives of the present work were thus to (i) study
the effect of biochar on N leaching through different soil layers
during the application of organic N fertilizer to agricultural soil in
layered columns; and (ii) to investigate the effect of biochar on soil
bacterial community structure under simulated leaching condition
via high-throughput sequencing method.
2. Materials and methods
2.1. Soil and biochar materials
Plow layer soil was collected from a farmland at the fluvo-aquic
soil test base of Chinese Academy of Agricultural Sciences,
Changping County, Beijing, China. The soil was air-dried and passed
through a 2 mm nylon sieve and mixed to get a homogeneous soil
sample before use. Corn straw (Zea mays L.) was oven dried (80  C)
and converted into biochar through slow pyrolysis using a furnace
(Olympic 1823HE) in a N2 environment at 500  C for 1.5 h. Biochar
samples were ground and sieved to get <1 mm sized particles.
Basic properties of the tested soil and corn straw biochar are
presented in Table 1. The soil had a high pH value due to the
presence of many coral limestone fragments that released calcium
ions. Compared to soil, biochar had a higher pH (10.0) and electrical
conductivity (EC, 1319 mS cm
1). The pH and EC of soil and biochar
(1:5 and 1:10 w/v, respectively) were measured in deionized water
using a pH meter (Mettler Toledo Delta 320) and an electrical
conductivity meter (DDS-307A), respectively. The concentrations of
soil ammonium and nitrate were determined using a flow injector
auto analyzer (Auto Analyzer 3, High Resolution Digital Colorim-
eter, Germany) in 1 M KCl extract (1:10 w/v) [17]. The CEC of soil
and biochar was measured with the ammonium-acetate
Table 1
Basic physiochemical properties of the tested soil and corn straw biochar.
Soil
pH 8.1
Organic matter (g kg
1) 16.4
Ammonium N (mg kg
1) 0.8
Nitrate N (mg kg
1) 5.7
Soil bulk density (g cm
3) 1.58
Cation exchange capacity (cmol kg
1) 17.4
Electrical conductivity (mS cm
1) 141.4
Field moisture capacity (%) 24.8
compulsory displacement method [18].
Ash content was determined by combusting the biochar at
750  C for 6 h in open crucibles on a dry weight basis. The carbon
(C), hydrogen (H), nitrogen (N) and oxygen (O) contents of biochar
were measured using an elemental analyzer (vario PYRO cube,
Germany). BrunauereEmmetteTeller specific surface area of the
biochar was determined using nitrogen gas on a Micrometrics ASAP
2010 system (Micrometrics, Norcross, GA, USA).
2.2. Leaching experiment
Layered soil column was constructed according to previous
methods [19,20] as depicted in Fig. S1. This device consisted of
three separated sections which were well joined and sealed
throughout the experiment. Soil column with dimension of 10 cm
inner diameter and 42 cm length were constructed with poly-
methyl methacrylate pipes and fitted with polyvinyl chloride end-
cap. There were three sampling openings on the column, i.e. a small
hole drilled on the sidewall at three different heights, representing
soil depths of 10, 20, and 30 cm along the soil profile. The sampling
opening was for extraction of soil leachate from different layers in
the profile. A 5-cm thick layer of coarse sand was placed at the
bottom of each column to further prevent soil loss. A simple water
container was used for supplying deionized water to the column to
simulate leaching conditions. About three kilograms of prepared
soil were packed into the columns to achieve an initial bulk density
of about 1.3 g cm
3. The top 10 cm soil in the column was subjected
to thorough mixing with 0.325 g urea (equal to 250 kg N ha
1) and
biochar with four different application rates of 0, 2, 4 and 8% (mass
ratio of biochar/soil, equivalent to 0, 40, 80 and 160 t ha
1), which
were designated as CK, B2, B4 and B8, respectively. Three replicates
were conducted for each treatment. The columns were kept in an
artificial greenhouse at 25 ± 2  C and a relative humidity of 65%.
Before starting the leaching experiment, about 1200 mL
deionized water was added from the top of each column over a
period of 7 days for the initiation of ammonification and nitrifica-
tion in soil. During the leaching period, deionized water was added
slowly into each column. Around 10 mL leachate was sampled from
the openings at 10, 20 and 30 cm depth along the soil column
respectively. Once the leachate volume approached 10 mL, the
addition of deionized water was suspended, and the total volume of
leachate was measured. All the sampling openings were sealed
when not sampling. Leachate was sampled at an interval of half a
month during the first three months and of one month during the
last two months for a total period of five months. The leachate
samples were filtered through disposable 0.45 mm pore-size filters
(Whatman, Clifton NJ, USA) and analyzed for pH, EC, nitrate,
ammonium and total N according to the methods mentioned
above.
Corn straw biochar
pH 10.0
C (%) 58.0
H (%) 2.7
O (%) 21.5
N (%) 2.3
Ash content (%) 16.7
Cation exchange capacity (cmol kg
1) 23.8
Electrical conductivity (mS cm
1) 1319
Specific surface area (m2 g
1) 14.7
 N. Xu et al. / European Journal of Soil Biology 74 (2016) 1e8
2.3. Soil microbial biomass, respiration rate and net N
mineralization
The soil in the first layer was sampled at the end of the exper-
iment. Soil microbial biomass, respiration rate and net N mineral-
ization were measured. Soil microbial biomass carbon (MBC) and
nitrogen (MBN) were measured using a chloroform fumigation-
direct extraction procedure [21,22]. The value to calculate
biomass from the C and N determinations (KEC and KEN) was 0.45
and 0.68 [21,23]. For each column, duplicate soil samples (20 g dry
weight equivalent) were weighed out. One was directly extracted
with 0.5 mol L
1 K2SO4 at a soil to solution ratio of 1:4. Another was
fumigated with chloroform for 24 h, followed by extraction with
the same K2SO4 solution. The concentrations of C and N in the
extracts were determined by an automated total organic carbon/
total nitrogen analyzer (Multi N/C, 3100/HT1300, Analytik Jena,
Germany).
Soil basal respiration (SBR) was measured at the end of the
experiment through incubation at the condition of 25  C and 60%
water holding capacity. The soil samples (20 g) were sealed in 1 L
jars, and CO2 was collected with 10 mL of 0.01 M NaOH solution
during a 4 h period. The resulted solution was then titrated with
HCl solution to determine the amount of absorbed CO2 [24]. Active
microbial biomass was measured using the substrate-induced
respiration (SIR) method [25]. Briefly, 1:4 glucose/talcum mixture
was added to the samples, then incubated under the same condi-
tions as for the basal respiration study, at a concentration of 12.0 g
glucose kg
1 soil. Respiration rates were reported as milligram CO2
per kilogram soil per day. As a method of assessing the efficiency of
microbial biomass, the metabolic quotient (qCO2) was calculated by
dividing SBR by MBC [25].
Net N mineralization was determined with a reported method
[26]. For this purpose, duplicate soil samples of 10 g dry weight
were taken from the top 10 cm layer of the column at the end of the
experiment. One was directly extracted with 1 mol L
1 KCl at a 1:10
of soil to solution ratio. Another was incubated in the dark at
constant humidity (45% of soil water holding capacity) and tem-
perature (25  C) for 15 days. At the end of the incubation, the
samples were subjected to extraction with the same extractant. N
concentrations in the extracts were determined, and net mineral-
ized N in soil was the difference in extracted N before and after
incubation.
2.4. DNA extraction, PCR-amplification of 16S rRNA gene fragments
and DNA sequencing
At the end of the experiment, soil samples were taken from the
top 10 cm layer of triplicate columns and mixed for bacterial
community analysis for each treatment. Total microbial DNA was
extracted from approximately 5 g soil with the MoBio PowerMax
Soil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA). DNA
concentrations were quantified using a NanoDrop ND-1000
UVeVis spectrophotometer (NanoDrop Technologies, USA).
The V3eV4 region of 16S rRNA gene was used as the bacterial-
specific fragment with the primers 338F (5 0 -ACTCCTACGGGAGG-
CAGCAG-3 0 ) and 806R (5 0 -GGACTACHVGGGTWTCTAAT-3 0 ) [27].
Error-correcting barcodes were added to both forward and reverse
primers [28]. PCR was conducted using a PTC 100 thermal cycler
(MJ Research, Waltham, MA, USA). All amplifications were per-
formed in 25 mL reactions with four replicates. Each reaction vol-
ume contained 1 mL of DNA template (about 20 ng), 0.5 mL of each
appropriate primer (at a final concentration of 0.2 mM), 0.25 mL of
bovine serum albumin (at a final concentration of 6 mM) (Takara,
Japan), 12.5 mL of 2  DreamTaq Green PCR Master Mix (Thermo
Scientific, USA). The following cycle parameters were used: initial
3
denaturation for 3 min at 95  C; 27 cycles of 30 s at 95  C, 30 s at
55  C, and 45 s at 72  C; and final extension for 10 min at 72  C. PCR
products were separated by gel electrophoresis, and fragments
with size in the range of 500e600 bp were excised from the gel and
extracted by using the Qiagen gel extraction kit (Qiagen, Valencia,
CA). Further purification was performed with Qiagen PCR purifi-
cation kit (Qiagen). Samples were pooled at equal concentrations.
Sequencing of the amplicons was performed by using the Illumina
HiSeq platform (Illumina, San Diego, CA, USA).
Raw sequences were classified with the Ribosomal Database
Project (RDP) training set using a confidence cut-off of 60% and
clustered into operational taxonomic units (OTUs) at 97% identity
with consensus taxonomy by single sequence analysis software the
Quantitative Insights into Microbial Ecology (QIIME 1.6.0) toolkit.
This generated a quality filtered dataset. The dataset was then
abundance filtered by removing OTUs with <20 counts across all
samples. Rarefaction curves were generated for quality-filtered and
quality plus abundance-filtered data sets to observe the sampling
efficiency. The internal complexity of individual sample was
calculated by a-diversity indices including Shannon-Weaver (H)
and Simpson index, Chao1, Ace and observed species. The multiple
samples similarity tree was constructed using an approximate
maximum likelihood method designed for large alignments as
implemented by the software FastTree.
2.5. Statistical analysis
The soil physicochemical and N leaching results were expressed
as means and standard deviations. Statistical analysis was per-
formed by using Statistical Product and Service Solutions 22.0 (SPSS
Inc., Chicago, IL, USA). Significant differences were obtained by the
one-way analysis of variance (ANOVA) with means compared using
the Duncan's multiple range test. The correlation was analyzed
with the Pearson test (two-tailed) at p ¼ 0.05. Any differences be-
tween the mean values at p < 0.05 were considered statistically
significant.
3. Results
3.1. Nitrogen leaching
The mass cumulative nitrate, ammonium and total N in the
leachates from all three layers under different treatments are
shown in Fig. 1. The nitrate, ammonium and total N leaching all
decreased under biochar treatments. The temporal changes of the
nitrate, ammonium and total N in leachates from different layers of
the soil columns under different treatments are shown in Fig. S2, S3
and S4, respectively. In particular, the difference in cumulative mass
of total N leaching between the control and biochar treatments was
more significant in the second layer as depicted in Fig. S4. The
change pattern of total N in leachates from different layers under
different treatments was similar to that of nitrate, as more than
ninety percent of total leached N was in the form of nitrate.
In detail, biochar significantly reduced the cumulative amount
of leached nitrate from all three layers by 16.0%, 16.7% and 19.3% in
the 2%, 4% and 8% biochar treatment, respectively (p < 0.05), as
compared to CK (33.6 mg, Fig. 1a). Similarly, the cumulative amount
of leached ammonium was decreased by 19.1%, 26.9% and 28.1%
with 2%, 4% and 8% biochar treatment, respectively (p < 0.05), as
compared to CK (0.9 mg, Fig. 1b). In all, biochar significantly
reduced the cumulative amount of total N leached by 18.8%, 19.5%
and 20.2% in the 2%, 4% and 8% treatment, respectively (p < 0.05), as
compared to the control (37.3 mg, Fig. 1c). This meant that
increasing amount of biochar resulted in reduced amount of N
leaching.
4 N. Xu et al. / European Journal of Soil Biology 74 (2016) 1e8
Fig. 1. Cumulative mass of (a) NO
þ
3 , (b) NH4 and (c) total N in the leachates from all three layers of the soil columns under different treatments (CK: no biochar; B2, B4 and B8 with
2%, 4% and 8% biochar/soil, respectively). Significant differences are indicated by different letters (p < 0.05).
As revealed in Fig. S5, in order to obtain the same amount of
leachate (around 10 mL) from each layer, the cumulative volume of
added water was 1276, 1380, 1421 and 1489 mL for CK, B2, B4 and
B8, increased by 8.1%, 11.4% and 16.7% as compared to the control
(p < 0.05), respectively. In addition, significant difference was also
observed under B8 as compared to B2 and B4 (p < 0.05).
3.2. Leachate pH and EC, soil microbial biomass, respiration rate
and net N mineralization
Figs. 2 and 3 depict the temporal change of pH and EC in the
leachates from different layers of soil columns under different
treatments, respectively. Leachate pH was higher under biochar
treatments, and increased with elevated amount of added biochar
in the first layer. At the end of the experiment, leachate pH in the
first layer increased by 0.21 unit on average under the biochar
treatments, compared to CK. Although leachate pH was still higher
in the second and third layers under biochar treatments, it was no
longer correlated with the biochar addition ratio. Unlike leachate
pH, leachate EC gradually increased with the elevated adding ratio
of biochar in all three layers, and increased along the soil profile
(p < 0.05) as depicted in Fig. 3. Compared to CK, leachate EC
increased by 46.7%, 48.8% and 79.5%, respectively, in the third layer
under 2%, 4% and 8% biochar treatments at the end of the
experiment.
Table 2 lists soil biochemical parameters and soil respiration rate
in the top 10 cm soil layer. Similarly, soil pH was increased in the
first layer under biochar treatments and had a positive correlation
with the addition ratio of biochar (p < 0.05). In terms of MBC and
MBN, significant difference was only observed between B8 treat-
ment and the control (p < 0.05). With 8% biochar treatment, MBC
increased by 10.8% in comparison to the control (75.1 mg kg
1), and
MBN increased by 7.5% as compared to CK (8.2 mg kg
1). SBR
8.4
a 10 cm
pH of leachate
8.0
7.6
7.2
0 15 30 45 60 75 90 105 120 135 150
Fig. 2. Temporal change of pH in the leachates from different layers of the soil columns under different treatments (CK: no biochar; B2, B4 and B8 with 2%, 4% and 8% biochar/soil,
respectively). Significant differences are indicated by different letters (p < 0.05).
increased by 32.3% on average as compared to the control (70.7 mg
CO2 kg
1 d
1) (p < 0.05), and significant difference between all the
biochar treatments and CK was observed. SIR increased by 28.7% on
average in comparison with the control (152.9 mg CO2 kg
1 d
1)
(p < 0.05). In addition, compared to B4 (214.6 mg CO2 kg
1 d
1), SIR
significantly decreased under B8 treatment (p < 0.05). Similar to
SBR, soil metabolic quotient also significantly increased under
biochar treatments (p < 0.05). Net N mineralization was also
significantly enhanced by 40.3%, 34.4% and 18.0% in the 2%, 4% and
8% treatment, respectively (p < 0.05), in comparison to the control
(1.3 mg kg
1 d
1). However, the net N mineralization decreased as
the addition ratio of biochar increased, especially for B8.
3.3. Soil bacterial community structure
After sequence filtering there were 108,900 high-quality se-
quences in total from all 4 samples. The average read length was
440 bp. The number of sequences per sample ranged from 25,624 to
28,395 with an average of 27,225. RDP Classifier was used to assign
these sequences to different OTUs with a 3% nucleotide cutoff. A
total of 5427 OTUs were recovered from the 4 samples. Table 3 lists
a-diversity indices, read numbers and coverages under different
treatments. The sampling coverage was more than 99% suggesting
almost complete sampling coverage of diversity within samples.
Bacterial diversity increased under biochar treatments in compar-
ison with the control, and had a positive correlation with the
addition ratio of biochar.
Fig. 4 showed the multiple samples similarity tree and relative
abundances of dominant bacterial phylum taxa in four different
treatments. Relative abundances of dominant bacterial genera in
four different treatments were depicted in Fig. S6. Relative abun-
dances of taxa at genera level in four dominant phyla detected in
different treatments were showed in Fig. S7. These results revealed
8.0
CK
B2
B4 b 20 cm a
a 7.6
ab c 30 cm
a b
B8 a a
a
b
b 7.6 b
b
7.2
7.2
6.8
0 15 30 45 60 75 90 105 120 135 150 0 15 30 45 60 75 90 105 120 135 150
Incubation time (d)
 N. Xu et al. / European Journal of Soil Biology 74 (2016) 1e8 5

1200 a 10 cm CK
B2
2400
b 20 cm
6000
c 30 cm

Electrical conductivity
of leachate (μs cm )
B4
5000

-1
1000 B8 2000

800 a 1600 4000

600 3000
b 1200
400 c a 2000
ab a
200 d 800 b b
b
c 1000 c
0 400
0 15 30 45 60 75 90 105 120 135 150 0 15 30 45 60 75 90 105 120 135 150 0 15 30 45 60 75 90 105 120 135 150

Incubation time (d)

Fig. 3. Temporal change of electrical conductivity in the leachates from different layers of the soil columns under different treatments (CK: no biochar; B2, B4 and B8 with 2%, 4%
and 8% biochar/soil, respectively). Significant differences are indicated by different letters (p < 0.05).

Table 2
Soil physicochemical parameters, soil respiration, and net N mineralization under different treatments (CK: no biochar; B2, B4 and B8 with 2%, 4% and 8% biochar/soil,
respectively). MBC ¼ microbial biomass carbon; MBN ¼ microbial biomass nitrogen; SBR ¼ soil base respiration; SIR ¼ substrate-induced respiration; qCO2 ¼ soil metabolic
quotient. Different letters in a single row indicate significant difference between the treatments at p < 0.05 (Duncan's multiple range test). Values are presented as
means ± standard deviation with n ¼ 3.

CK B2 B4 B8

pH 7.84 ± 0.18c 8.17 ± 0.20b 8.21 ± 0.14a 8.25 ± 0.12a

MBC (mg kg
1) 75.12 ± 4.63b 79.45 ± 4.27ab 75.31 ± 4.21b 83.27 ± 5.01a
MBN (mg kg
1) 8.24 ± 0.31b 8.56 ± 0.50ab 8.59 ± 0.42ab 8.86 ± 0.30a
SBR (mg CO2 kg
1 d
1) 70.68 ± 5.23b 89.19 ± 5.76a 96.86 ± 6.31a 94.53 ± 5.89a
SIR (mg CO2 kg
1 d
1) 152.95 ± 12.81c 186.41 ± 10.35b 214.58 ± 10.41a 189.51 ± 9.58b
qCO2 (d
1) 0.94 ± 0.04b 1.12 ± 0.11a 1.29 ± 0.12a 1.14 ± 0.03a
Net N mineralization (mg kg
1 d
1) 1.28 ± 0.19c 1.80 ± 0.19a 1.72 ± 0.15a 1.51 ± 0.12b

Table 3
Comparison of a-diversity indices, read numbers and coverages under different treatments at a genetic distance of 3% (CK: no biochar; B2, B4 and B8 with 2%, 4% and 8%
biochar/soil, respectively).

Treatment Read numbers Coverages OTU Chao1 Ace ShannoneWeaver

CK 19,693 0.992028 1279 1361 1357 6.27

B2 19,557 0.992126 1327 1402 1401 6.29
B4 20,989 0.992186 1401 1493 1477 6.36
B8 21,097 0.992795 1420 1498 1487 6.44

Fig. 4. Multiple samples similarity tree (left) and relative abundances of dominant bacterial phyla in four different treatments (right) (CK: no biochar; B2, B4 and B8 with 2%, 4% and
8% biochar/soil, respectively).

that bacterial community structure was different between biochar different treatments, with relative abundances ranging from 22.9%
treatments and the control. Biochar treatments with high addition to 8.6% and decreased gradually under biochar treatments. Relative
ratios (B4 and B8) have higher similarity as depicted in Fig. 4. abundances of Gemmatimonadetes and Chloroflexi were also
The dominant phyla observed across all treatments included decreased with biochar addition. On the contrary, Proteobacteria,
Proteobacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Actino- Bacteroidetes and Actinobacteria were increased after biochar
bacteria, Nitrospirae and Gemmatimonadetes. These taxa accounted application. Number of sequences classified to be within six
for more than 85% of the bacterial sequences in all the four treat- ammonium oxidizing bacteria (AOB) genera under four treatments
ments (Fig. 4). Acidobacteria was the most sensitive phylum under is shown in Table 4. Although there was no obvious difference
6 N. Xu et al. / European Journal of Soil Biology 74 (2016) 1e8
Table 4
Number of sequences classified to be within the six ammonia oxidizing bacterial
genera under four treatments (CK: no biochar; B2, B4 and B8 with 2%, 4% and 8%
biochar/soil, respectively).
Gene CK B2 B4
Nitrolancea 4 4 0 3
Nitrosomonas 7 9 23
Nitrosospira 30 161 201
Nitrospira 1121 893 1090
Nitrosomonadaceae_uncultured 976 507 1010
Nitrospinaceae_uncultured 11 17 23
between biochar treatments and CK in the total number of AOB
sequences, biochar addition changed the relative abundance of
these AOB genera. In particular, the relative abundance of Nitro-
sospira obviously increased under biochar treatments.
4. Discussion
4.1. Effect of biochar on N leaching
It has been reported that enhanced nitrogen retention in the
biochar-treated soil was related to increased soil aggregation
resulting in higher water holding capacity [6]. Soil aggregation was
dependent on the number of pores and pore size distribution as
well as the specific surface area of soil [6]. As revealed in Fig. S5, in
order to obtain the same amount of leachate (around 10 mL) from
each layer, more water addition was needed for the columns with
biochar treatments. This could be due to the high porosity of bio-
char. As more water was added under biochar treatments, nitrogen
concentration in the leachate declined as depicted in Fig S2, S3 and
S4.
Urea is transformed to ammonium bicarbonate when applied in
soil which is a natural process resulting from the activity of urease
enzyme. Then the ammonium is generally converted to nitrate
through nitrification [29]. With the addition of biochar in soil, the
nitrate, ammonium and total N leaching all decreased. Nitrate
leaching was about one order magnitude greater than ammonium,
while the cumulative mass of leached ammonium from all three
soil layers was less than 1 mg in all treatments. The result was
consistent with the findings of previous work [30] in which
ammonium sulfate was applied in a short-term lysimeter study.
The results suggested that ammonium leaching from agricultural
soil was not a major problem because it was readily adsorbed onto
negatively charged clay minerals in soil. Compared to nitrate
leaching, there had a significant difference between the lowest
addition ratio of biochar (B2) and higher addition of biochar
treatments (B4 and B8) in terms of the cumulative mass of leached
ammonium (p < 0.05). The results indicated the stronger adsorp-
tion of free NHþ 4 on biochar particles due to the relatively higher
CEC of biochar (23.8 cmol kg
1) in comparison with soil
(17.4 cmol kg
1) [31].
4.2. Effect of biochar on leachate pH and EC, soil microbial biomass,
respiration rate and net N mineralization
The potential liming effect of biochar has been reported [32].
Decarboxylation of organic anions (i.e. ash alkalinity) of added
biochar consumes protons thus the soil pH and leachate pH both
increased. The increment in leachate EC could be attributed to the
ash content in biochar (16.7% by mass) which contained an amount
of mineral ions, such as calcium, magnesium, potassium and so-
dium. As the mineral ions dissolved and leached in water, leachate
EC was increased under biochar treatments.
Microbial biomass carbon and nitrogen had no significant in-
crease under B2 and B4 treatments. No significant change of mi-
crobial biomass was also observed under low biochar addition ratio
(<8%) in temperate soil [33]. However, some other studies showed
B8 significant increase of soil microbial biomass under low biochar
application rate (<2%) [8,34]. These discrepant results may be
5 explained in part by the variation of biochar (i.e. biochar feedstock,
120
pyrolysis temperature, etc.) and soil types. Both soil and biochar
1220
1074 were alkaline in the present study, and pH has been reported as one
9 of the driving parameters for any effect on soil microbial biomass,
community, and activity [5].
Higher respiration rates for biochar amended soil could have
been mediated by an improved soil structure, leading to enhanced
aeration and microbial activity [35]. The significant increment of
soil metabolic quotient under biochar treatments (p < 0.05) was
consistent with a previous study [24] in which the value of qCO2
was also suggested as an indicator of bioenergetic status of mi-
crobial biomass.
The increment in net N mineralization under biochar treatments
could be due to the labile C in biochar which enhanced soil mi-
crobial activity thereby influenced N transformation [36]. Similarly,
enhanced net N mineralization was observed in the soil amended
with N fertilizer and manure biochar [37].
These results showed that biochar addition could improve soil
microbial activity which could be due to the labile C in biochar and
more available nitrogen in soil under biochar treatments. In turn,
higher microbial activity indicated possibly higher turnover ratio of
nutrient such as nitrogen thereby N leaching may be reduced under
biochar treatments. However, it should be noted that SIR of B8
(189.5 CO2 kg
1 d
1) significantly decreased (p < 0.05) as compared
to B4 (214.9 CO2 kg
1 d
1), and net N mineralization of B8
(1.7 mg kg
1 d
1) also significantly decreased (p < 0.05) as
compared to B4 (1.51 mg kg
1 d
1), suggesting that there are other
factors which could slow down microbial activity in soil at the
higher biochar application rate, such as heavy metals in biochar
[38].
4.3. Effect of biochar on soil bacterial community structure
It has been suggested that biochar may affect soil bacterial
community via improving soil physicochemical properties [9].
Combining the results of Figs. 1e4 and Table 2, biochar addition
increased soil pH and EC, decreased N leaching, increased microbial
biomass and shifted the bacterial community composition. These
results were consistent with the previous report in which the
biochar impacts on soil microbial community composition was
investigated in an acidic soil [9]. In addition, because of the high
porosity and various functional groups, biochar may build up
biogeochemical interfaces (BGIs). The compositional heterogeneity
of BGIs could diversify the niche microhabitats thus support growth
of highly diverse bacterial communities [39]. Thereby the bacterial
diversity would be higher with more added biochar. This could be
one reason why B4 and B8 treatments have higher similarity while
the control was different from all the biochar treatments in terms of
bacterial diversity as depicted in Fig. 4.
As to the bacterial community composition, the relative abun-
dance of Acidobacteria, Chloroflexi and Gemmatimonadetes
decreased under biochar treatments, while Proteobacteria, Bacter-
oidetes and Actinobacteria increased. This could be due to syner-
gistic effects, such as co-metabolism or syntrophy, and/or similar
response patterns to biological, chemical or physical variables, i.e.
occupation of similar niche space [10]. The phylum Proteobacteria
was the most predominant taxa in all soil samples, which was
known to colonize nutrient-rich environments [40].
It has been reported that Actinobacteria are often associated
 N. Xu et al. / European Journal of Soil Biology 74 (2016) 1e8
with the degradation of recalcitrant polymers and thus considered
to be ecologically important in the turnover of organic matter in
soil [41]. The higher presence level of Actinobacteria in biochar-
amended soil in comparison with CK may be a consequence of
their ability to degrade recalcitrant carbon compounds [42].
Generally, biochar amendment was characterized by an increase in
the relative abundance of Actinobacteria [11,14], as confirmed in the
present study.
Previous studies suggested that Acidobacteria played an impor-
tant role in biogeochemical cycling of carbon and consequently
might be adaptable to the environment of large variety of carbon
sources present in biochar [42]. However, the soil showed a more
alkaline environment after biochar amendment, which is not
favorable for Acidobacteria, a phylum of bacteria usually being
acidophilic [43]. As a result, the abundance of Acidobacteria
decreased with biochar addition. The relative abundance of Gem-
matimonadetes declined possibly because they prefer drier soils
[44].
The phylum Nitrospirae was responsible for biogeochemical
cycling of nitrogen and was accessible to the large amount of ni-
trogen resources added in the form of urea. Unlike the previous
study [45] in which very few sequences (<100 reads) were shown
to belong to AOB in forest soils using pyrosequencing method, a
considerable number of sequences from the AOB genera were
observed in the present study. This could be due to the fertilized
agricultural soil (and applied with 250 kg N ha
1) used which has
higher nitrogen content in comparison with forest soil. As the
genera Nitrosospira was also reported to play an important role in
the emission of nitrous oxide [46], the increment of its sequence
under biochar treatments suggested that biochar may increase
greenhouse gas emission [47]. These results indicated that the
composition of N cycling-related bacterial community was changed
under biochar treatments, thus the microbial transformation of N
could have been influenced by biochar which may be another
mechanism for reduced N leaching [6].
5. Conclusions
Simulated nitrogen leaching experiments were conducted with
the application of organic N fertilizer (urea) in layered soil columns.
Due to the biological transformation of urea into ammonium and
then nitrate, and the strong sorption of ammonium in soil, more
than ninety percent of total N leaching was in the form of nitrate.
Total N leaching was reduced by 18.8%, 19.5% and 20.2% under the
application of 2%, 4% and 8% biochar treatment in comparison with
the control, respectively (P < 0.05). The relative abundance of
Acidobacteria, Chloroflexi and Gemmatimonadetes decreased under
biochar treatments, while Proteobacteria, Bacteroidetes and Actino-
bacteria increased. Soil microbial biomass slightly increased, while
soil bacterial diversity was significantly higher, which may have
contributed to the transformation of organic N fertilizer and
reduction of N leaching.
Acknowledgments
This study was financially supported by the Special Fund for
Agro-scientific Research in the Public Interest (201303095-10), the
National Natural Science Foundation of China (41301311), and the
Open Fund of Key Laboratory of Nonpoint Source Pollution Control,
Ministry of Agriculture, China (2014-37).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejsobi.2016.02.004.
7
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